Spectroscopic imaging can be used to determine the spatially distributed and chemically distinct species in heterogenous materials. Spectroscopic imaging is an analytical tool that has been applied at both the macroscopic and microscopic levels.
At the macroscopic level, chemical imaging of areas exceeding 20 square kilometers can be achieved using airborne and spaceborne remote sensing, near-infrared imaging spectrometers. On a smaller scale, diagnostic imaging of the human body is possible through nuclear magnetic resonance imaging techniques (MRI).
At the microscopic level, fluorescence imaging is a technique employed for chemical state microscopy. Fluorescence is the emission of radiation (light) through which a molecule in an electronically excited state is able to dissipate its excess energy. Molecular fluorescence emission normally occurs at visible wavelengths. Fluorescent intensities from fluorophores (analyte molecules with fluorescent properties) which exhibit high quantum efficiencies can be relatively strong. Fluorescence spectroscopy, in general, is an extremely sensitive technique. In fact, single molecule detection has been demonstrated utilizing fluorescent spectroscopy. Fluorescence microscopy involves the labeling of a specific component of interest with a fluorescent tag and the subsequent viewing of the spatially resolved fluorescence emission. Improved specificity can be provided by immuno-fluorescent tags in which the analyte is an antigen that binds to a fluorescently tagged antibody. Many types of fluorescent immunoassays have been developed and are widely used in biomedical and biological imaging. Additional background information on fluorescence microscopy can be found in "Applications of Fluorescence in the Biomedical Sciences," D. L. Taylor, A. S. Waggoner, R. F. Murphy, F. Lanni, R. R. Birge (eds.), Liss, New York (1986).
Another class of spectroscopic microscopy techniques employs vibrational spectroscopy. Specifically, the methods of Raman and infrared spectroscopy provide chemical selectivity without requiring undue sample preparation or incurring sample alteration or degradation. Raman spectroscopy, an inelastic light scattering phenomenon, is commonly used to characterize a sample on the basis of its molecular vibrational spectrum. The Raman scattering phenomenon is observed by illuminating a sample with a high intensity monochromatic source, such as a laser, and detecting the fraction of light scattered at longer wavelengths. Typically, about 1 Raman photon is scattered per 10.sup.8 incident photons. The frequency displacements of the Raman scattered light from the incident laser radiation correspond to the vibrational frequencies of the sample molecules. Since the vibrational spectral bandwidths observed in Raman spectroscopy are usually 5-30 cm.sup.-1 (0.3-6 nm at 750 nm), spectroscopic devices must be able to resolve, as well as detect, these faint signals.
Traditionally, Raman spectroscopy has been performed using visible wavelength lasers, optics and detectors coupled to monochromators that employ diffraction gratings for spectral dispersion and isolation. More recently, laser-referenced Michelson interferometers have been employed in conjunction with solid-state near-infrared (NIR) laser excitation, primarily Nd:YAG, for Fourier transform (FT) Raman spectroscopy. Additional information can be found in "Fourier-Transform Raman Spectroscopy of Biological Materials," Anal Chem., 62(21) 1990, Ira W. Levin and E. Neil Lewis. One advantage of FT-Raman spectroscopy is the improved instrumental performance of FT interferometers over standard monochromators. Specifically, the instrument provides intrinsic throughput (Jacquinot's advantage) and multiplex (Fellgett's advantage) characteristics, as well as high spectral precision (Connes' advantage) and the capacity for high spectral resolution. Commercial FT interferometer systems can typically provide 0.02-5 cm.sup.-1 (0.02-5 nm at 3300 nm) spectral resolution. Current interferometer based instruments are optimized for maximum signal throughput, but are designed without regard for maintaining image fidelity through the device. An FT interferometer that retains image fidelity would provide the inherent advantages of interferometry and could also be suitable for spectroscopic imaging.
Infrared (IR) spectroscopy involves the absorption of IR radiation, generally between 770.degree.-10000.degree. nm (12,900.degree.-10.degree. cm.sup.-1), by molecular species. Energies in the infrared region of the spectrum are on the order of the energies of vibrational transitions, and IR spectroscopy is complementary in its information content to Raman spectroscopy. IR spectroscopic imaging is applicable to a wide range of materials, but is especially well suited to the study of polyatomic organic molecules, as vibrational frequencies are well correlated with organic functional groups. In particular, IR and Raman spectroscopy are suitable for the study of biological materials. Almost all materials absorb infrared radiation, except homonuclear diatomic molecules (O.sub.2, H.sub.2, N.sub.2). Polyatomic molecules exhibit rich IR spectra. The spectra include both the fundamental absorptions in the mid-IR (2500.degree.-20000.degree. nm), but also the overtones and combination bands, primarily of O--H, C--H and N--H absorptions, in the near-IR (770-2500 nm). While the near-IR bands are significantly weaker than the fundamental bands, the wavelengths at which they are observed are compatible with quartz and germanium refractive optics, making the near-IR region of the spectrum well suited for high spatial resolution chemical state imaging studies based on molecularly specific vibrational absorptions.
The sensitivity of Raman and infrared spectroscopy to even small changes in molecular structure is well established, and these techniques are capable of generating specific fingerprints for a given molecular species. In general, systems capable of generating infrared absorption or Raman emission images find wide use in a variety of areas in science and technology. Materials amenable to these types of analysis would include, but not be limited to biological materials, polymers, superconductors, semiconductors and minerals.
Currently, two primary methods are employed for image generation. The first approach involves the systematic scanning of a sample. Typically, this is achieved either by translating the sample through a stationary field of view defined by the collection optics and detector, or alternatively, by scanning the imaging source (or detector) in a raster pattern across the surface of the stationary sample. The scanning approach is typically utilized with a single element detector. An example of a vibrational spectroscopic imaging device employing the scanning method is a Fourier transform infrared (FTIR) spectrometer coupled to a mid-infrared microscope outfitted with an x-y mapping translation stage for imaging a sample. The technique utilizes the infrared (vibrational) absorption properties of molecular functional groups in the sample to generate the image.
Scanning methods for vibrational spectroscopic imaging, while workable, have certain drawbacks and deficiencies. Specifically, the signal to noise ratios obtainable with FTIR microspectrometers often requires substantial signal averaging at each spatial position, thus making the FTIR systems inherently slow. As a result, only crude spatial maps are generally obtained. In addition, the near-infrared imaging spectrometers employed for remote sensing typically use diffraction gratings for spectral characterization which require that images be constructed a slice at a time as the spectrometer scans the sample surface. Furthermore, the numerous moving parts contained within these systems limit the speed and reliability of these devices.
A second method of image generation involves wide field illumination and viewing in conjunction with multichannel detection. Direct viewing with a color video camera of a subject illuminated by a broadband visible source is a simple example of wide field illumination imaging. In such a case, colorometric information based on the visible absorption of the sample is obtained.
For greater specificity and selectivity, fluorescence microscopy may be performed. In fluorescence microscopy, optical filtering of an intense arc lamp illumination source to select strong plasma lines can be employed to selectively excite a molecular fluorescent label added to a sample. Alternatively, a laser is employed for illumination having a wavelength output which falls within the absorption range of the fluorescent label and selectively excites the tag. The fluorescent light, which emits at longer wavelengths than the excitation source, is commonly discriminated using dielectric interference filters. Where several spectral regions are to be viewed separately, filter wheels containing multiple filters can be utilized. Fluorescent spectral linewidths are usually 10-100 nm wide. Where only a single type of fluorophore is present in a sample, spectral filters providing relatively broad spectral resolution, 5-25 nm, can be adequate. Where multiple similar fluorophores are present simultaneously, multiple filters providing spectral resolution of 1-2 nm may be necessary.
Wide field illumination methods employing glass or interference filters, however, have certain drawbacks and deficiencies. Specifically, the application of discrete notch filters for spectral selectivity requires the use of a separate filter at each desired wavelength, ultimately limiting operation to only several wavelengths. In addition, the dielectric notch filters employed provide resolution of approximately 10-100 nm, which is often an inadequate spectral resolution for discriminating similar but different species in multicomponent environments. The techniques using filter wheels provide only limited spectral resolution and spectral coverage, and also suffer from the constraints of moving mechanical parts, limiting the speed and reliability of these systems.
A hybrid spectroscopic imaging method combining wide field illumination and multichannel detection with spatial multiplexing has been developed. The technique is called Hadamard transform spectroscopic microscopy and has been especially adapted for Raman emission microscopy. Additional information can be found in "Multichannel Hadamard Transform Raman Microscopy," Appl. Spectrosc., 44(2) 1989, Patrick J. Treado and Michael D. Morris. The technique employs a dispersion spectrograph as the spectral filter and is capable of generating spectral images of a variety of materials at sub-micron spatial resolution.
The multichannel/spatial multiplex method, however, also has certain limitations. Specifically, the number of spectral features that can be collected simultaneously is determined by the inherently limited spectral coverage of the spectrograph. Where survey spectral images are to be collected, the Hadamard imager is not optimal. In addition, artifacts can arise in the spectral images due to systematic spatial encoding errors. These artifacts ultimately compromise the spatial resolution of the technique.